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D Mangoura

Publications and source records attributed to D Mangoura.

At least 37 records · Page 2Linked to original sources
PubMed1993

Large chondroitin sulfate proteoglycans of developing chick CNS are expressed in cerebral hemisphere neuronal cultures.

Chondroitin sulfate proteoglycans (CSPG) of the extracellular matrix may play regulatory roles in central nervous system (CNS) development. We have examined the expression of two large CSPGs of the embryonic chick brain, which can be differentiated using the monoclonal antibodies HNK-1 and S103L, in cultures of embryonic day 6 chick cerebral hemisphere neurons. Western blot analysis following immunoprecipitation and endoglycosidase treatment revealed that these cultures produce S103L- and HNK-1-reactive proteoglycans which are biochemically indistinguishable from the CSPGs (previously) identified in homogenized chick embryo brain extracts. The HNK-1-reactive CSPG accumulated in the medium throughout the course of cultures. In contrast, the S103L-reactive CSPG was found in a neuron-associated form during the period of aggregate establishment in culture, as well as in a soluble form secreted into the medium. Immunocytochemical staining of cultures with the S103L antibody localized reactivity to most neurons during the period of aggregate formation, while neuronal processes and the few flat cells present (presumably neuroblasts and early glia) were negative. Cell selection experiments confirmed that neurofilament-positive cells were the source of the S103L-reactive CSPG. The use of differential fixation techniques suggested that the cell-associated S103L reactivity may be intracellular. Because of this pattern of expression and localization, we propose that the developmentally regulated S103L-reactive CSPG may play a role in neuronal migration arrest and organization of neurons into functional aggregates.

Animals↗
PubMed1993

Opioid peptides activate phospholipase D and protein kinase C-epsilon in chicken embryo neuron cultures.

The mu-opioid peptide morphiceptin stimulated a Ca(2+)-independent protein kinase C (PKC-epsilon) that is expressed both in embryonic day 6 chicken telencephalon and in derived neuronal cultures. This activation was seen as a 2-fold increase in the activity and level of cytosolic PKC-epsilon and as a transient increase in membrane-associated PKC-epsilon following morphiceptin treatment. Morphiceptin did not activate phospholipase C-mediated phosphatidylinositol hydrolysis but did transiently activate (2- to 3-fold) phospholipase D (PLD), as measured by phosphatidylethanol formation in neuron cultures derived from embryonic day 6 or day 7 cerebral hemispheres. This PLD activation could provide an alternative source of diacylglycerol for the activation of PKC-epsilon and was naloxone-reversible and at least partially blocked by the tyrosine kinase inhibitor herbimycin A. Addition of phorbol 12-myristate 13-acetate stimulated both PLD and PKC-epsilon activities to a greater extent than opioids. The phorbol ester and insulin stimulation of PLD was also blocked by herbimycin. Both morphiceptin (in a naloxone-reversible manner) and phorbol ester increased phosphorylation of similar cytosolic proteins in intact cells, demonstrating a functional role for the PKC-epsilon activation by opioids. This is evidence that opioid receptors are transiently coupled to tyrosine kinase, PLD and PKC-epsilon activation and, by implication, to neuronal cell growth during brain morphogenesis.

Analgesics↗
PubMed1991

Chronic opioid treatment attenuates carbachol-mediated polyphosphoinositide hydrolysis in chick embryo neuronal cultures.

Opiate binding sites on cultured neurons derived from 6-day-old (E6) chick embryo cerebral hemispheres (CH), shown to be cholinergic by choline acetyltransferase immunostaining, were labeled with [3H]etorphine (mu and delta opiate receptors expression) and [3H]morphine (mostly mu). When examined by light microscope autoradiography, opiate receptors were found to be expressed by most neurons, and were distributed predominantly on neuronal perikarya. Muscarinic and opiate receptors in E6CH cultured neurons were found to be functionally coupled when the effects of opiate receptor occupancy on the inositol phosphate-linked muscarinic receptors was studied. Carbachol stimulated the release of [3H]inositol phosphates (InsP) from cultures preincubated with [3H]inositol and LiCl, in a dose-dependent manner, and the functional expression of muscarinic receptors peaked in number at day 7 in culture, declining thereafter. Short-term (less than 1 h) treatment of E6 neuronal cultures with 1 microM opioid peptides such as morphiceptin or D-Ala2-D-Leu5-enkephalin (DADLE) did not inhibit the release of inositol phosphates in response to 1 mM carbachol whereas forskolin, which also activates adenylate cyclase and raises cAMP levels, inhibited InsP release by about 25%. In contrast, long-term (48 h) opioid treatment with either morphiceptin or DADLE (1-10 microM) inhibited the carbachol-stimulated inositol phosphate release by greater than or equal to 50%. Prolonged treatment with morphiceptin also inhibited the bradykinin-mediated release of InsP from E6CH cells. In both cases, the inhibition was partially blocked by the continuous presence of naloxone, suggesting that the inhibition was mediated through opiate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗
PubMed1990

Evidence for plasticity in neurotransmitter expression in neuronal cultures derived from 3-day-old chick embryo.

We have previously reported the developmental profiles of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT) bio- and immunocytochemically, assessing GABAergic and cholinergic neuronal phenotypes respectively, in neuroblast-enriched cultures from 3-day-old chick embryo, plated on poly-L-lysine. We have also reported that collagen as culture substrate inhibits neuronal aggregation and neuritic fasciculation in this culture system. In this study we assessed the same parameters for cultures on collagen. In addition, we evaluated the effects of nerve growth factors (NGF) on cholinergic and GABAergic expression on neurons plated either on polylysine or collagen. We found that non-neuronal cells and NGF prolonged the survival of cholinergic and GABAergic neuronal populations and that both markedly stimulated GABAergic expression. In contrast, cholinergic expression was only enhanced by NGF. Immunostaining for GABA and ChAT reflected the biochemical findings. Glutamine synthetase and cyclic nucleotide phosphohydrolase, used as markers for astrocytes and oligodendrocytes respectively, showed very low activity in both substrata and were not related to GAD or ChAT peak activities. Our findings suggest that humoral factors and cell-cell contacts markedly influence neuronal phenotypic expression in culture. Moreover, it appears that during early neuronal differentiation GABAergic neurons are more responsive to microenvironmental regulation compared to cholinergic neurons.

Animals↗
PubMed1989

Developmental profile of glutamine synthetase in lines of mice bred for ethanol sensitivity.

Glutamine synthetase (GS) activity was used as a marker to examine differences in astrocyte development in mice selectively bred for ethanol sensitivity: long sleep (LS), short sleep (SS), mild ethanol withdrawal (MEW), severe ethanol withdrawal (SEW) and control ethanol withdrawal (CEW). We found that 1) GS activity in MEW and SEW was higher than in LS and SS during the first 2 weeks of postnatal development, in the forebrain but not in the cerebellum; 2) lower GS activity was observed consistently in all areas examined with the SS mice as compared to the LS; 3) glutamine synthetase activity in MEW and SEW differed significantly from their controls (CEW) during the early developmental period regardless of the brain region examined; however, after 30 days of maturation, GS activity in SEW was higher than that in MEW and CEW in the forebrain. Astrocytes are known to contribute in the regulation of the neuronal microenvironment. Therefore, we interpret the differences we found in astrocytic function during early brain development among these lines of mice to account in part for the neuronal predisposition to ethanol sensitivity.

Aging↗
PubMed1989

Early and late passage C-6 glial cell growth: similarities with primary glial cells in culture.

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell number and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into "astrocytic phenotype". Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.

Animals↗
PubMed1988

GABAergic neurons in cultures derived from three-, six- or eight-day-old chick embryo: a biochemical and immunocytochemical study.

Cultures were prepared from 3-, 6- and 8-day-old chick embryos. Whole chick embryos were dissociated by sieving through a nylon mesh for E3 cultures and cerebral hemispheres for E6 and E8 cultures. The dispersed cells were plated onto poly-L-lysine coated culture dishes in Dulbecco's modified Eagle's medium, containing 10% fetal bovine calf serum. Growth patterns in these cultures have been previously described. Biochemical and immunocytochemical markers were used to identify GABAergic neurons in culture. Neurons exhibiting GABA-like immunoreactivity were present in all 3 types of cultures as early as 4-6 days in culture. The developmental profile of glutamic acid decarboxylase (GAD) derived from 3-day-old whole chick embryo cultures showed low activities whereas the enzyme activity markedly rose in cultures derived from 6- or 8-day-old chick embryo cerebral hemispheres during the first two weeks. The changes in GAD activity observed in these cultures are interpreted to reflect the maturational state of GABAergic neurons and also their responsiveness to microenvironmental factors.

Animals↗
PubMed1988

Cholinergic neurons in cultures derived from three-, six- or eight-day-old chick embryo: a biochemical and immunocytochemical study.

Cultures were prepared by dissociating 3-day-old whole chick embryos, or 6- or 8-day-old chick embryo cerebral hemispheres. The dispersed cells were plated onto poly-L-lysine coated dishes in Dulbecco's modified Eagle's medium fortified with 10% fetal bovine calf serum. Immunocytochemistry, histochemistry and biochemistry were used to characterize the cholinergic neurons in these cultures. Double staining for acetylcholinesterase (AChE) and neurofilament revealed that a high percentage of neurons also contained AChE; fewer neurons though were positive for choline acetyltransferase (ChAT). The presence of AChE in non-cholinergic neurons was interpreted to be indicative of 'cholinoceptive' neurons in culture. The developmental profile for ChAT activity in cultures derived from 3-day-old whole chick embryos was previously described; we have reported that activity reached high levels by 7 days in culture. In this study we found that in cultures derived from 6-day-old chick embryos ChAT activity increased slowly up to 6 days in culture and rose markedly thereafter; in contrast, in cultures derived from 8-day-old chick embryos activity was low until day 9 and rose moderately thereafter. The differences in ChAT expression between cultures derived from chick embryos of different developmental stages may reflect a loss of plasticity of the cholinergic population with age.

Acetylcholinesterase↗
PubMed1988

Factors influencing neuronal growth in primary cultures derived from 3-day-old chick embryos.

We compared neuronal growth patterns in primary cultures prepared by dissociating 3-day-old chick embryos, either whole embryo (E3WE) or head only (E3H) and plating the dispersed cells onto Petri dishes coated with either poly-L-lysine, collagen or laminin. The culture medium was Dulbecco's Modified Eagle's Medium (DMEM), supplemented with either 5 or 10% fetal bovine calf serum (FCS). As we have previously described, in E3WE cultures on poly-L-lysine the neuronal primary growth patterns were aggregation with neuritic fasciculation, presence of growth cones with microspikes and very few flat cells. In contrast with cultures grown on poly-L-lysine, in cultures grown on collagen or laminin the distinct growth pattern was extensive networks of isolated and differentiated neurons lying on acquired monolayers of flat cells. When 5% FCS was used, as compared to 10% FCS, neuronal aggregates were fewer and smaller on poly-L-lysine; on collagen or laminin a tendency to aggregate was observed. Several differences were observed in the E3H cultures when compared to E3WE: (a) aggregates were less numerous with the prevailing pattern being a web-like, self-contained aggregate; (b) aggregates connected with other aggregates or flat cells were rare and the aggregate adhesivity was minimized; (c) neurons on collagen or laminin formed networks with the exception of a few, small aggregates displaying no fasciculation; (d) flat cells did not form a monolayer but islets which hosted the neuronal meshy networks. We attribute these differences in the growth patterns between the various types of cultures to be the combined result of a variety of environmental signals, derived from the provided substrata, the serum and the nonneuronal cell factors and cell surface, all primarily regulating neuronal adhesivity.

Animals↗
PubMed1986

Growth patterns of glial cells dissociated from newborn and aged mouse brain with cell passage.

Glial cell cultures derived from newborn and aged (18-month-old) mouse cerebral hemispheres and maintained up to cell passage 11 were characterized immunocytochemically by using glial fibrillary acidic protein (GFA), and biochemically by using glutamine synthetase (GS), for astrocytes, and 2',3' cyclic nucleotide 3' phosphohydrolase (CNP) for oligodendrocytes. We report here the changes occurring during passages 5-11. GS and CNP activities did not significantly change with cell passage in cultures from newborn mouse. In cultures derived from aged mouse, CNP activity did not change significantly whereas GS activity increased severalfold. A characteristic finding in higher cell passages (passage #7) was the loss of GFA-positive stained cells and the appearance of multinucleated cells. We interpret these changes in culture to represent possible signs of cellular senescence.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1986

Growth patterns of primary cultures dissociated from 3-day-old chick embryos: morphological and biochemical comparisons.

Cultures were prepared by dissociating 3-day-old whole chick embryos and plating the dispersed cells on poly-L-lysine-coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts between neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell population was characterized histochemically by the method of Karnovsky and Roots and biochemically by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase-positive staining; however, with days in culture, several aggregates had no staining, and some positive-stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.

Acetylcholinesterase↗
PubMed1986

Effects of neuron-conditioned medium and fetal calf serum content on glial growth in dissociated cultures.

The influence of the microenvironment as assessed by medium conditioned by 6-day-old chick embryo neurons in culture and of the nutrients derived from fetal bovine serum was evaluated in cultures of primary chick embryo glial cells. Glia-enriched cultures from 15-day-old chick embryo were incubated from culture days 3-9 with various concentrations of neuron-conditioned medium, with or without 10% fetal bovine serum in the final culture medium. Also, glial growth was studied in cultures with 5%, 10% or 20% fetal bovine serum in the medium. Glutamine synthetase and 2',3',-cyclic nucleotide 3'-phosphohydrolase were used as astrocytic and oligodendrocytic markers, respectively. Cultures were harvested at day 9. The presence of neuron-conditioned medium in the cultures was associated with persistence of immature glioblast-like cells. This persistence of glial immature cells was also reflected by the lower glutamine synthetase activity in the cultures with neuron-conditioned medium as compared to cultures with neuron-conditioned medium and fetal calf serum. In cultures with 5% neuron-conditioned medium without fetal bovine serum, cyclic nucleotide phosphohydrolase activity was increased. We are assuming that the input of neurons to the microenvironment is partially mediated through the neuron-conditioned medium. Thus, the present findings show that neurons influence the growth and differentiation of glial cells in culture.

Animals↗
PubMed1986

Effects of opiates on the growth of neuron-enriched cultures from chick embryonic brain.

Neuron-enriched cultures derived from 6-day-old chick embryo cerebral hemispheres were treated with morphine or methadone, 10(-5) M or 10(-6) M, on days 4-6 or 6-8 in culture and were evaluated morphologically and biochemically at day 9 using phase contrast microscopy and choline acetyltransferase activity (ChAT) as a cholinergic marker. The treatment of the cultures with morphine markedly affected their growth pattern; specifically, we observed an increased number of flat cells presumptively glia, and aggregates sided by flat cells and devoid of thick bundles of neuritic processes that normally characterize neuron-enriched cultures. These morphologic changes were reflected in a drastic decrease of ChAT activity in cultures treated from day 4 to day 6 but not from 6 to 8. In contrast to morphine, exposure to 10(-6) M methadone from day 4 to day 6 resulted in reduced ChAT activity but the growth pattern of the cultures remained morphologically intact. We suggest that morphine exerts a general neurotoxic effect whereas methadone may affect some specific cholinergic function.

Animals↗
PubMed1984

Glial cells dissociated from newborn and aged mouse brain.

Changes occurring with days in culture and cell passage in cultured glial cells derived from newborn vs aged (18-mo) mouse cerebral hemispheres were compared. The activities of the enzymes glutamine synthetase (GS), an astrocyte marker, and 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNP), an oligodendrocyte marker, were determined. In addition, glial fibrillary acidic protein (GFA) and glycerol phosphate dehydrogenase (GPDH) immunoreactivity was used to morphologically identify astrocytes and oligodendrocytes, respectively. In cultures derived from newborn mouse cerebral hemispheres, both GS and CNP activity and GFA-positive and GPDH-rhodamine-positive cells were present with cell passage. In general, GS activity did not change in early cell passage in cultures from either newborn or aged mouse; in passage 5, GS was high in both sources of cell populations. CNP activity increased with cell passage in cultures derived from newborn mouse; in cultures derived from aged mouse CNP was low in the primary cultures, increased with cell passages 2 and 3, and declined with passages 4 and 5. The survival of astrocytes as shown by GS and the decline in oligodendrocytes as shown by CNP was also supported by an increase in the proportions of GFA and GPDH immunoreactive cells. We interpret the increase in GS activity to parallel the astrogliosis observed in vivo in the aging brain. Moreover, the decline in oligodendrocytes in culture may represent a shift of balance between glial cell types that appears to be influenced by the age of brain tissue and time in culture.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1984

Glial cell growth in culture: influence of living cell substrata.

The role of the microenvironment in the growth of glial cells in culture has been the topic of ongoing research in this laboratory. Recently, we reported a study on the contribution of fibroblast cell substratum and extracellular matrix in glial cell growth. In the present study we report data concerning a) the influence of a neuronal-enriched living substratum from chick embryo on the growth of glial cells derived from chick embryonic brain and plated onto the substratum; b) the influence of dissociated cells derived from chick embryonic brain on the growth of established glial cells in culture, and c) the influence of dissociated cells derived from adult rat spinal cord on the growth of established glial cells from newborn rat in culture. The activities of glutamine synthetase (GS) and 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP) were the biochemical probes determined for astrocytes and oligodendrocytes, respectively. We found that glial growth as assessed by both enzyme activities, was enhanced when a nervous tissue derived cell population was plated onto a glial-enriched substratum, whereas glial growth was inhibited when the neuronal-enriched population was the cell substratum.

Age Factors↗